The long-term goal of this project is to define the molecular mechanisms which regulate the activity of the mammalian sirtuin, SIRT6, especially in the context of genome stability. Additionally, the studies outlined in this proposal will delineate the relevance of these regulatory controls in regards to aging and the onset of age- related disease. SIRT6 has emerged as a critical regulator of multiple pathways related to aging, including DNA repair, telomere maintenance, tumorigenesis, inflammation and glycolysis. Moreover, it has been demonstrated that SIRT6 overexpression extends the lifespan of mice. Despite the overwhelming evidence that SIRT6 operates at the crux of multiple pathways related to aging, very little is known about the upstream regulatory mechanisms which control the activity of SIRT6 and allow it to regulate such a diverse array of cellular processes. In this application we propose to identify the regulatory pathways that control the activity of SIRT6; in particular, we will focus on understanding the mechanisms that regulate the activity of SIRT6 in the context of genome stability. Recent studies by our laboratory demonstrated that SIRT6 is an upstream regulator of DNA double strand break (DSB) repair. We showed that SIRT6 stimulates both pathways of DSB repair under oxidative stress. Our unpublished preliminary data shows that SIRT6 is phosphorylated by JNK1/2 in response to oxidative stress on amino acid S10 and that this phosphorylation is required for the stimulation of DSB repair. We have also shown that, in addition to controlling DSB repair, SIRT6 maintains genome stability by repressing transposable elements, and that oxidative stress causes re-localization of SIRT6 from the promoters of transposable elements to the sites of newly formed DNA breaks. Thus, we are ideally positioned to conduct further mechanistic studies of SIRT6 regulation in the context of genome stability. As such, we will pursue the following specific aims: (1) identify the mechanisms which regulate the ability of SIRT6 to stimulate DNA repair in response to oxidative stress; (2) identify the mechanisms which regulate the ability of SIRT6 to suppress expression of LINE-1 retrotransposons; and (3) determine the role of SIRT6 phosphorylation in genome stability and longevity by constructing mouse models with mutations in the SIRT6 phosphorylation sites. The proposed research will provide novel and important insights into the regulatory mechanisms, which govern SIRT6 activity as well as delineate new pathways regulated by SIRT6 which are relevant to genome stability and aging. As such, we expect that these experiments will reveal critical, new information about the aging process, and will help to develop novel strategies for treating age-related diseases, in particular diseases of genome instability such as cancer.